Reference genes for RT-qPCR normalisation in different tissues, developmental stages and stress conditions of Hypericum perforatum

Abstract

Abstact14 Hypericum perforatum is a widely known medicinal herb used mostly as a remedy for depression 15 because of its abundant secondary metabolites. Quantitative real-time PCR (qRT-PCR) is an 16 optimized method for the efficient and reliable quantification of gene expression studies. In general, 17 reference genes are used in qRT-PCR analysis because of their known or suspected housekeeping 18 roles. However, their expression level cannot be assumed to remain stable under all possible 19 experimental conditions. Thus, the identification of high quality reference genes is very necessary for 20 the interpretation of qRT-PCR data. In this study, we investigated the expression of fourteen 21 candidate genes, including nine housekeeping genes and five potential candidate genes. Additionally, 22 the HpHYP1 gene, belonging to the PR-10 family associated with stress control, was used for 23 validation of the candidate reference genes. Three programs were applied to evaluate the gene 24 expression stability across four different plant tissues, three developmental stages and a set of abiotic 25 stress and hormonal treatments. The candidate genes showed a wide range of Ct values in all 26 samples, indicating that they are differentially expressed. Integrating all of the algorithms and 27 evaluations, ACT2 and TUB-β were the most stable combination overall and for different 28 developmental stages samples. Moreover, ACT2 and EF1-α were considered to be the two most 29 applicable reference genes for different tissues and for stress samples. Majority of the conventional 30 housekeeping genes exhibited better than the potential reference genes. The obtained results will 31 contribute to improving credibility of standardization and quantification of transcription levels in 32 future expression research of H. perforatum. 33 37 [1][2][3][4]. Methods for detecting gene expression include northern blot, gene chips, semi-PCR, RNase 38 protection analysis and qRT-PCR. qRT-PCR because of its high sensitivity, specificity, 39 reproducibility and accuracy has become a very popular and effective method for detecting and 40 quantifying gene transcription levels [5][6][7]. Reliable quantification of gene expression levels by qRT-41 PCR analysis requires the standardization and fine-tuning of several parameters, such as the amount 42 of initial sample, RNA recovery and integrity, enzymatic efficiency of cDNA synthesis and PCR 43 amplification, and the overall transcriptional activity of the tissues or cells analysed [5][6][7][8][9]. The 44 expression stability of frequently used reference genes also cannot be neglected. Therefore, 45 normalization for transcript levels of test genes is essential for minimizing technical differences for 46 different samples and experimental conditions [9]. More importantly, selection of stable reference 47 gene(s) is prerequisite before qRT-PCR analysis.48 A appropriate reference gene is considered to be not affected by the experimental conditions and also 49 shouldh maintained at a invariable levle amon...